Certain proteins synthesized by animals, insects, plants and unicellular eukaryotes such as yeast are glycosylated; that is, oligosaccharide chains are covalently attached to the polypeptide backbones. These glycoproteins are primarily localized on the external surface of the plasma membranes of cells and on the lumenal face of intracellular organelles such as lysosomes of the Golgi apparatus. They are also secreted into extracellular fluids.
The carbohydrates moieties of the glycoproteins are believed to be involved in a variety of biological functions, influencing both the biological activity of individual glycoproteins as well as inter- and intracellular recognition events. The carbohydrate units of a particular glycoprotein can modulate the solubility of the protein, protect it from heat denaturation of proteolytic degradation, control its circulatory lifetime, directs its site of uptake in an animal, and influence its antigenicity.
The carbohydrate moieties attached to proteins exposed on the external face of the plasma membrane of cells are believed to be important determinants for biological recognition. They serve as cell surface receptors for certain growth factors, hormones, toxins, viruses, bacteria, and lectins. Cell surface carbohydrates are also involved in the interactions of cells with the extracellular matrix and with other cells.
In view of the known and postulated biological functions of the oligosaccharides of glycoproteins, these carbohydrate units have become the subject of intense interest and experimental investigation. Studies of the carbohydrates include efforts to analyze structure, to determine routes of biosynthesis and catabolism, to examine the specificity of individual enzymes in the metabolic pathways, to modulate the biological activity of glycoproteins by alteration of their carbohydrates, to attach carbohydrates to proteins, and, of course, to determine their biological functions.
Free reducing oligosaccharides obtained from natural sources, and derivatives of the oligosaccharides, have been used extensively for such investigations. For most structural studies, it is desirable to introduce a reporter group such as a radioactive, fluorescent, or UV-active chromophore at the reducing end of the oligosaccharide chain. The most common procedures to accomplish this are reduction with NaB[.sup.3 H].sub.4 (S. Takasaki and A. Kobata, Meth. Enzymol, 50, 50-54 (1978)) or reductive amination with 2-aminopyridine and sodium borohydride. (S. Hase et al., J. Biochem. (Tokyo), 95, 197-203 (1984)).
The resulting products and similar derivatives are also useful as substrates for studies of the interaction of carbohydrate chains with receptors.
Free reducing oligosaccharides or their derivatives have also been coupled through the reducing sugar to other materials for a variety of purposes. Examples include coupling of the oligosaccharide to a resin for use as an affinity ligand, coupling to a carrier protein in order raise antibodies against carbohydrate, and coupling to a protein to alter the protein's biological activity. Potential improvements in the biological activity of a protein include an increase in solubility, an increase in stability to proteolytic degradation or heat denaturation, a reduction in antigenicity or immunogenicity, an increase or decrease in serum lifetime, or a targeting to a specific cell type in the body such as hepatocytes or macrophages.
Many strategies have been employed for coupling carbohydrates to proteins or other substrates (Y. C. Lee and R. T. Lee, The Glycoconjugates, Vol. IV, M. I. Horowitz (Ed.), Academic Press, N.Y., 1982, pp 57-83; J. D. Aplin and J. C. Wriston, Jr. CRC Crit. Rev., 10, 259-306 (1981)). These include, but are not limited to, diazo coupling, thiocarbamylation, amidation, amidination, reductive alkylation, transglutamination and the use of bifunctional reagents such as 2,4,6-trichloro-s-triazine and 2,4-diisocyanotoluene.
The reactions used to prepare the various oligosaccharide derivatives generally result in reduction of the pyranose ring to the open chain (alditol) form or in formation of an O- or S-glycosidic linkage at the reducing end of the oligosaccharide. These derivatization reactions can be difficult to carry out when working with oligosaccharides derived from Asn-linked glycan chains.
The present invention is concerned with enzymatic methods for obtaining 1-amino-1-deoxyoligosaccharides from peptides and proteins containing Asn-linked oligosaccharides and converting them to N-glycoside derivatives that preserve the beta-glycosylamine linkage and the pryanosyl ring of Asn-linked glycoproteins.